SUMMARY: PROJECT 5 (POSTMORTEM STUDIES OF PACAP-CRF IN HUMAN PTSD/BERRETTA) Compelling evidence indicates that corticotropic releasing factor (CRF) and pituitary adenylyl cyclase-activating polypeptide (PACAP), as well as their interactions together, make critical contributions to stress responses, anxiety, circadian rhythm regulation, and the pathophysiological mechanisms of post-traumatic stress disorder (PTSD). The underlying neural circuitry involved is poorly understood, but important clues point to the bed nucleus of the stria terminalis (BNST), amygdala (AMG), dorsal anterior cingulate gyrus (dACG), and the hypothalamus (HPTh) as critical regulators of these functions. Current evidence indicates that CRF and PACAP mechanisms that contribute to PTSD may be sex-specific, raising the possibility that the underlying brain changes and potential therapeutic targets may differ in males and females. Current and preliminary data suggest that PACAP signaling may directly affect CRF expressing cells and that circadian expression of PACAP and its cognate receptor PAC1R, and their subsequent regulation of CRF systems, may vary during the course of the day. Such variations may potentially contribute to disruptions of sleep/wake cycles associated with DSM-defined illnesses including PTSD, major depression, and anxiety disorders. Surprisingly, virtually no information is available on cell-level expression of CRF and PACAP signaling pathways in the human AMG, BNST, dACG and HPTh, their relationships to circadian rhythms, and the involvement of CRF/PACAP interactions in the neuropathology of PTSD. Our overarching hypothesis is that abnormalities affecting CRF/PACAP pathways in the BNST, AMG and dACG and HPTh contribute to the pathology of PTSD. In Aim 1, we address a critical gap of knowledge on the region-, sex- and circadian- specific expression and distribution of CRF, PACAP, and their receptors in healthy human brain. Our hypothesis is that protein and mRNA expression of CRF, PACAP, and their receptors are region- and sex-specific; in particular, we predict that PACAP receptors will show cell- and sex- specificity and expression in CRF-positive neurons, supporting the hypothesis that PACAP regulates these neurons in a sex dependent manner. In Aims 2 and 3, we examine whether?at the protein, gene expression, and cellular level?signaling pathways in the dACG, AMG, BNST and HPTh are altered in PTSD. Our hypothesis is that CRF and PACAP signaling pathways will be altered in subjects with PTSD, relative to healthy controls, with increased PACAP expression correlated with CRF signaling pathway changes, in a region-, sex-, and circadian rhythm-specific manner. We predict that increases of PACAP-positive cells and axons, reflecting increased PACAP expression locally and from hypothalamic inputs, will be accompanied by altered expression of PACAP receptors and down-stream signaling pathways in CRF-positive cells. Project 5 may identify CRF and PACAP systems and circuits as being fundamentally altered in stress-related illnesses such as PTSD, and is a key nexus of the Center that enhances, and is enhanced by, the other (preclinical, clinical) elements.